Cobalt Regulates Activation of Camk2α in Neurons by Influencing Fructose 1,6-bisphosphatase 2 Quaternary Structure and Subcellular Localization

Abstract

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD⁺ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co²⁺, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co²⁺, and we provide a line of evidence that Co²⁺ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca²⁺/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.

Keywords: Fbp2; mitochondria; moonlighting protein; protein-protein interaction.

Figure 1

Co²⁺ stimulates Fbp2 association with mitochondria but it does not alter the nuclear accumulation of the protein. (A) Fbp-mitochondria colocalization is significantly increased in Co²⁺-treated neurons both in the presence and in the absence of Fbp inhibitor (iFbp). In the presence of Co²⁺, the amount of mitochondria-bound Fbp is as high as after the LTP induction. The iFbp-treated neurons show reduced colocalization compared to control cells. (B) Co²⁺ treatment does not change the nuclear accumulation of Fbp in neurons.

Figure 2

Co²⁺ does not modulate oligomerization of Fbp2 but it affects the AMP-bound protein migration in the Native-PAGE. (A) Size-exclusion chromatography (SEC) reveals that in a presence of AMP and Co²⁺, Fbp2 adopts tetrameric conformation. As a control, we used the Fbp2 L190G mutein which cannot tetramerize and adopts dimeric form only. (B) Migration of the Co²⁺-saturated and Co²⁺-free Fbp2 in the Native-PAGE is similar. In the absence of ligands, Fbp2 migrates mainly as the R-state tetramers, although the small amount of dimers is also visible (black line). The dimeric arrangement is the only confirmation for the Fbp2 L190G mutein (red line). The mobility of the AMP-induced T-state Fbp2 (magenta line) is increased as compared to the R-state. However, in the simultaneous presence of Co²⁺ and AMP, the mobility of Fbp2 (blue line) does not fully reflect that observed in the presence of AMP alone.

Figure 3

Quantification of Fbp2 protein in Co²⁺-treated neurons. (A) Fbp-related fluorescent signal in neurons incubated with Co²⁺ is significantly higher than in control cells, whereas the fluorescence is lower in iFbp-treated cells. The decrease in Fbp fluorescence is not observed in neurons treated simultaneously with iFbp and Co²⁺. (B) Western blot analysis demonstrates that Fbp2 protein amount does not differ significantly among control neurons, neurons incubated with Co²⁺, and with Co²⁺/iFbp mixture. Tetramerization of Fbp2 with iFbp reduces the protein-associated signal in comparison to control neurons.

Figure 4

The effect of Co²⁺ on Fbp2 kinetics. (A) Co²⁺ acts as an Fbp2 inhibitor in the presence of catalytic ions (Mg²⁺) with the maximal inhibition of about 79% and IC₅₀ value 6.1 µM, whereas (B) in the absence of Mg²⁺, Co²⁺ activates Fbp2 with the AC₅₀ value of 8.6 µM. (C) Co²⁺ acts in a cooperative manner competing with Mg²⁺ for binding to the active site of Fbp2. (D) Comparison of the Fbp2 enzymatic activity in the presence of Co²⁺, AMP, and both the agents.

Figure 5

The effect of Co²⁺ on the amount of total and phosphorylated Camk2α in neurons. The fluorescence related to total Camk2α (A) and pThr286 Camk2α (B) is significantly elevated in neurons incubated with Co²⁺ and Co²⁺/iFbp, whereas in the cells treated with iFbp alone, the signal related to total Camk2α decreases and pThr286 Camk2α-related fluorescence is unaltered. (C) Western blot and its densitometric analysis (D) reveals that the total amount of the kinase is increased only in Co²⁺-treated neurons, whereas the level of pThr286 Camk2α is higher in Co²⁺- and Co²⁺/iFbp-incubated neurons.

Figure 6

The Fbp-Camk2α complex formation occurs on mitochondria. (A,B) The effect of Co²⁺ and iFbp on Fbp2-Camk2α complex formation in the context of mitochondrial localization. Co²⁺ significantly increased the complex-related signal while iFbp decreases it. (C) The complex is almost exclusively localized to mitochondria.

Figure 7

The effect of short-term Co²⁺-treatment of neurons. Incubation of neurons with Co²⁺ for 1 h resulted in (A) the increased Fbp-mitochondria colocalization and (B) elevated amount of detected pThr286 Camk2α. (C) The increased pThr286 Camk2α level after acute Co²⁺ treatment is not observed in neurons with partially silenced Fbp2 expression.